Project Summary Tobacco use is the leading cause of cancer. Menthol cigarettes make up approximately 27% of the total cigarette market in the United States and over 80% of African American smokers use menthol cigarettes. Menthol cigarettes are more difficult to quit than conventional cigarettes. Menthol has a neurobiological effect on the nicotinic acetylcholine receptors (nAChRs). At micromolar concentrations, menthol acts as a negative allosteric modulator of the ?4?2 nAChR, the most abundant nAChR in the brain and the one that plays the largest role in nicotine addiction. At nanomolar concentrations applied for over 24 hr., menthol causes the upregulation of ?4* and ?6* nAChRs in midbrain dopaminergic neurons. Most importantly, menthol enhances nicotine reward through its ability to enhance dopamine neuron excitability. Ultimately, studying the effects, functions, and mechanisms of chronic and acute menthol has tremendous clinical relevance. Recently, I found that incorporating a L9?A mutation into the second transmembrane helix (TM2) in the ?4?2 nAChR causes menthol to dramatically lose function as a negative allosteric modulator. This was observed whether the L9?A mutation was incorporated in the ?4 or ?2 subunit. Spurred by these results, I want to determine how acute menthol is functioning as an allosteric modulator. By investigating other nAChRs, using other residues at the 9? position, and mutating other sites on TM2, I will determine how menthol acutely effects the nAChRs. These experiments will utilize noncanonical amino acid mutagenesis and two-electrode voltage clamp electrophysiology in Xenopus laevis oocytes to elucidate this chemical mechanism. Additionally, nanomolar menthol over the course of at least 24 hr. upregulates the ?4* and ?6* nAChRs in midbrain dopaminergic neurons. The Lester lab is a leading contributor to the field of ?inside-out pharmacology? and menthol?s effects on ?4* and ?6* nAChRs falls under this category. I am interested in whether this effect is specific to menthol and ?4* and ?6* nAChRs or if chronic application of other cooling agents could elicit this response or if other ion channels are affected by chronic exposure to cooling agents. Total internal reflection fluorescence (TIRF) microscopy will be used to observe ion channel concentration on the cell surface as a measure of upregulation. Finally, the way menthol upregulates ?4* and ?6* nAChRs is not clear. We know that menthol acts on the endoplasmic reticulum exit sites (ERES) and on the cycling between the Golgi and the endoplasmic reticulum, but what is the first event affected by menthol is not known. The mechanism for nicotine-induced upregulation have been probed extensively by the Lester lab and I will do the same for menthol. By using RNA sequencing, biorthogonal noncanonical amino acid tagging, mass spectrometry, and particle tracking microscopy, I hope to find the earliest step in the nAChR exocytic pathway that is affected by menthol.